Mark Groudine

Comprehensive mapping of long range interactions reveals folding principles of the human genome

Chromosomal Mapping The conformation of the genome in the nucleus and contacts between both proximal and distal loci influence gene expression. In order to map genomic contacts, Lieberman-Aiden et al. (p. 289, see the cover) developed a technique to allow the detection of all interactions between genomic loci in the eukaryotic nucleus followed by deep sequencing. This technology was used to map the organization of the human genome and to examine the spatial proximity of chromosomal loci at one megabase resolution. The map suggests that the genome is partitioned into two spatial compartments that are related to local chromatin state and whose remodeling correlates with changes in the chromatin state. Chromosomes are organized in a fractal knot-free conformation that is densely packed while easily folded and unfolded. We describe Hi-C, a method that probes the three-dimensional architecture of whole genomes by coupling proximity-based ligation with massively parallel sequencing. We constructed spatial proximity maps of the human genome with Hi-C at a resolution of 1 megabase. These maps confirm the presence of chromosome territories and the spatial proximity of small, gene-rich chromosomes. We identified an additional level of genome organization that is characterized by the spatial segregation of open and closed chromatin to form two genome-wide compartments. At the megabase scale, the chromatin conformation is consistent with a fractal globule, a knot-free, polymer conformation that enables maximally dense packing while preserving the ability to easily fold and unfold any genomic locus. The fractal globule is distinct from the more commonly used globular equilibrium model. Our results demonstrate the power of Hi-C to map the dynamic conformations of whole genomes.

Controlling the double helix

Chromatin is the complex of DNA and proteins in which the genetic material is packaged inside the cells of organisms with nuclei. Chromatin structure is dynamic and exerts profound control over gene expression and other fundamental cellular processes. Changes in its structure can be inherited by the next generation, independent of the DNA sequence itself.

An expansive human regulatory lexicon encoded in transcription factor footprints

Regulatory factor binding to genomic DNA protects the underlying sequence from cleavage by DNase I, leaving nucleotide-resolution footprints. Using genomic DNase I footprinting across 41 diverse cell and tissue types, we detected 45 million transcription factor occupancy events within regulatory regions, representing differential binding to 8.4 million distinct short sequence elements. Here we show that this small genomic sequence compartment, roughly twice the size of the exome, encodes an expansive repertoire of conserved recognition sequences for DNA-binding proteins that nearly doubles the size of the human cis–regulatory lexicon. We find that genetic variants affecting allelic chromatin states are concentrated in footprints, and that these elements are preferentially sheltered from DNA methylation. High-resolution DNase I cleavage patterns mirror nucleotide-level evolutionary conservation and track the crystallographic topography of protein–DNA interfaces, indicating that transcription factor structure has been evolutionarily imprinted on the human genome sequence. We identify a stereotyped 50-base-pair footprint that precisely defines the site of transcript origination within thousands of human promoters. Finally, we describe a large collection of novel regulatory factor recognition motifs that are highly conserved in both sequence and function, and exhibit cell-selective occupancy patterns that closely parallel major regulators of development, differentiation and pluripotency.

Control of c-myc regulation in normal and neoplastic cells

Publisher Summary This chapter discusses normal c-myc gene regulation and abnormal c-myc regulation in cancer cells. All normal c-myc transcription units are composed of three exons: the second two encoding the major c-myc proteins. These two exons have from 70% to over 90% sequence identity between species. All c-myc genes contain a long untranslated exon 1, suggesting an important function for this feature. Other members of the myc oncogene family, N-myc and L-myc, share the three-exon gene organization with exons 2 and 3 providing the major coding regions that exhibit highly conserved stretches of amino acids. A long untranslated exon 1 is present in both N-myc and L-myc genes. These exons have little homology to each other or to exon 1 of c-myc, lending further support to the notion of an important structural or regulatory role for c-myc leader regions via a sequence-independent mechanism. Ample direct and circumstantial evidence exists to implicate c-myc in neoplastic transformation. Indirect evidence is provided by the presence of the c-myc gene at various DNA rearrangements that characteristically accompany tumors, such as leukemias, lymphomas, and small-cell lung carcinomas. These rearrangements may lead to one or more of increased levels, constitutive synthesis, or alterations in ratios between the c-myc products. Mutations in c-myc protein coding regions occur, but are not characteristic of rearranged c-myc in tumor cells.

Intragenic DNA methylation alters chromatin structure and elongation efficiency in mammalian cells

Transcriptional silencing in mammals is often associated with promoter methylation. However, a considerable number of genomic methylated CpGs exist in transposable elements, which are frequently found in intronic regions. To determine whether intragenic methylation influences transcription efficiency, we used the Cre/loxP-based system, RMCE, to introduce a transgene, methylated exclusively in a region downstream of the promoter, into a specific genomic site. This methylation pattern was maintained in vivo, and yielded a clear decrease in transgene expression relative to an unmethylated control. Notably, RNA polymerase II (Pol II) was depleted exclusively in the methylated region, as was histone H3 di- and trimethylated on Lys4 and acetylated on Lys9 and Lys14. As the methylated region adopts a closed chromatin structure in vivo, we propose that dense intragenic DNA methylation in mammalian cells initiates formation of a chromatin structure that reduces the efficiency of Pol II elongation.

Tissue-specific DNA cleavages in the globin chromatin domain introduced by DNAase I

Abstract Using recombinant chicken DNA clones as probes, we have investigated the DNAase I sensitivity of chromosomal DNA regions bordering the α- and β-globin structural genes. By both a blot hybridization assay and solution hybridization, we find that regions around these globin genes are preferentially sensitive (relative to the ovalbumin gene) to DNAase I after mild digestion of isolated red cell nuclei. These regions are resistant in cells that do not express globin. The preferential DNAase I sensitivity extends to at least 8 kb on the 3′ side of the β-globin gene cluster and to 6 or 7 kb on the 5′ side, where relatively resistant DNA fragments have been identified. Using low levels of DNAase I to titrate the sensitivities of coding and adjacent noncoding regions, it was observed that coding regions are organized into a very sensitive structure, while adjacent noncoding regions are organized into a moderately sensitive structure. The blot hybridization assay has also revealed that DNAase I introduces specific, double-stranded cuts into both the α- and β-globin gene clusters. Many of these cuts are tissue-specific. Several α gene-specific sites occur toward the 3′ side of the α-coding sequences. The β sites are different in embryonic and adult red cells. In embryonic cells, the cut occurs near the 5′ end of an embryonic β gene, while in adult cells there are two cuts, one at approximately 2 kb and the other at approximately 6 kb from the 5′ side of an adult gene. Based on the observation that the general region around the origin for replication and promotors for transcription in the SV40 minichromosome is also very sensitive to specific, double-stranded scissions by DNAase I, we speculate that the specific cuts in the globin domain may be structural modifications of the chromatin that are associated with origins for DNA replication or promotors for transcription.

An encyclopedia of mouse DNA elements (Mouse ENCODE)

To complement the human Encyclopedia of DNA Elements (ENCODE) project and to enable a broad range of mouse genomics efforts, the Mouse ENCODE Consortium is applying the same experimental pipelines developed for human ENCODE to annotate the mouse genome.

Genome-wide DNA replication profile for Drosophila melanogaster : a link between transcription and replication timing

Replication of the genome before mitotic cell division is a highly regulated process that ensures the fidelity of DNA duplication. DNA replication initiates at specific locations, termed origins of replication, and progresses in a defined temporal order during the S phase of the cell cycle. The relationship between replication timing and gene expression has been the subject of some speculation. A recent genome-wide analysis in Saccharomyces cerevisiae showed no association between replication timing and gene expression. In higher eukaryotes, the limited number of genomic loci analyzed has not permitted a firm conclusion regarding this association. To explore the relationship between DNA replication and gene expression in higher eukaryotes, we developed a strategy to measure the timing of DNA replication for thousands of genes in a single DNA array hybridization experiment. Using this approach, we generated a genome-wide map of replication timing for Drosophila melanogaster. Moreover, by surveying over 40% of all D. melanogaster genes, we found a strong correlation between DNA replication early in S phase and transcriptional activity. As this correlation does not exist in S. cerevisiae, this interplay between DNA replication and transcription may be a unique characteristic of higher eukaryotes.

Developmental and species-divergent globin switching are driven by BCL11A

The contribution of changes in cis-regulatory elements or trans-acting factors to interspecies differences in gene expression is not well understood. The mammalian β-globin loci have served as a model for gene regulation during development. Transgenic mice containing the human β-globin locus, consisting of the linked embryonic (ε), fetal (γ) and adult (β) genes, have been used as a system to investigate the temporal switch from fetal to adult haemoglobin, as occurs in humans. Here we show that the human γ-globin (HBG) genes in these mice behave as murine embryonic globin genes, revealing a limitation of the model and demonstrating that critical differences in the trans-acting milieu have arisen during mammalian evolution. We show that the expression of BCL11A, a repressor of human γ-globin expression identified by genome-wide association studies, differs between mouse and human. Developmental silencing of the mouse embryonic globin and human γ-globin genes fails to occur in mice in the absence of BCL11A. Thus, BCL11A is a critical mediator of species-divergent globin switching. By comparing the ontogeny of β-globin gene regulation in mice and humans, we have shown that alterations in the expression of a trans-acting factor constitute a critical driver of gene expression changes during evolution.

Nuclear compartmentalization and gene activity

The regulated expression of genes during development and differentiation is influenced by the availability of regulatory proteins and accessibility of the DNA to the transcriptional apparatus. There is growing evidence that the transcriptional activity of genes is influenced by nuclear organization, which itself changes during differentiation. How do these changes in nuclear organization help to establish specific patterns of gene expression?